Purification studies on medium conditioned by pokeweed mitogen-stimulated spleen lymphocytes (SCM) have shown that the active factor able to stimulate stem cell self-renewal is the glycoprotein Multi-CSF (IL-3). An inducible T-lymphocyte cell line (LB3) has been developed that synthesizes large amounts of Multi-CSF. The cDNA for Multi-CSF has been cloned from an mRNA library constructed from LB3 cells. The action of cloned Multi-CSF will be analyzed on the self-renewal of stem cells in three cell systems--normal marrow stem cells, Multi-CSF-dependent continuous multipotential cell lines, and v-ras-transformed hemopoietic cells. Studies will be continued on the mechanism of action of G-CSF in suppressing stem cell self-renewal in the myelomonocytic leukemia WEHI-3B, and attempts will be made to clone the receptor for G-CSF on these cells. (M) R01 CA26038 05 MGN KOEFFLER, H PHILLIP Tumor Biology UNIVERSITY OF CALIFORNIA LOS ANGELES 01/01/84 - 12/31/84 LOS ANGELES CA FUTURE YEARS: 3 DIFFERENTIATION AND PROLIFERATION OF MYELOID CELLS $94,254 Myeloid differentiation will be investigated using regulated complementary DNA (cDNA) clones of the HL-60 myeloid cell line, that is, clones that represent mRNAs that change in abundance as HL-60 differentiates. Of special interest will be the study of the molecular modulation of lysozyme and myeloperoxidase (MPO) expression. The human MPO and lysozyme cDNA clones will be isolated, a restriction endonuclease map of the genomic genes determined, and the genes assigned to precise chromosomal locations. The role of DNA methylation and chromatin structure in myeloid differentiation will be examined. The molecular defect(s) of hereditary and secondary MPO deficiency in humans will be studied. The common and divergent pathways of gene expression will be examined as HL-60 differentiates from promyelocytes to granulocytes and from promyelocytes to macrophage-like cells. Myeloid proliferation will be examined by studying the interaction of colony-stimulating factor (CSF) with myeloid cells. We shall use human myeloid leukemia clones and lines with different responses to CSF. One of the principal questions will be: does CSF responsiveness of the cell correlate with number and/or affinity of CSF receptors on the cell? Detection of leukemia transforming gene(s) from cells of myeloid leukemic patients and lines will be pursued using the NIH3T3 transfection assay. Is a different transforming gene(s) active in myeloid leukemic cells that are blocked at different stages of differentiation or have different karyotypic abnormalities? Do preleukemic cells have detectable transforming gene(s)? The planned studies should provide a better understanding of the control of proliferation and differentiation in acute myelogenous leukemia and, in a broader perspective, the control of normal cellular growth and differentiation. (M)